Dextrans and Pullulans - ACS Publications

21 Dextrans and Pullulans: Industrially Significant

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α-D-Glucans ALLENE JEANES Northern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Peoria, IL 61604 This symposium on extracellular microbial polysaccharides of practical importance would not be complete without consideration of the α-D-glucans, dextran and pullulan. The significance of dextran in man's practical affairs was apparent before i t s origin, identity, and name were established. Dextrans develop naturally in sucrose-containing solutions that have become inoculated with dextran-producing bacteria from a i r , plants, or s o i l . The resulting transformation of the solutions to syrupy, viscous, or ropey fluids, or even to gelled masses, doubtlessly has plagued man since the inception of accumulating and storing sucrose-containing foods and beverages. As early as 1813 (1,*2*) , r eports described the mysterious thickening or solidification of cane and beet sugar juices, and later impediment of filtration and crystallization was traced to the occurrence of this condition. In 1861, Louis Pasteur (3) initiated systematic scientific progress by explaining that these "viscous fermentations" resulted from microbial action. In 1878, van Tieghem (4) named the causative bacteria Leuconostoc mesenteroides because i t s growth in colorless flocs resembled that of the green algae of the genus Nostoc. In 1880 (5), Scheibler.established this type of product as a glucan having positive optical rotation and named it dextran. Thus, through the importance of the dextran class of α -D-glucans in man's economy, the dextrans were the f i r s t extracellular microbial polysaccharides to come under systematic scientific investigation. Dextrans from several bacterial strains also were the f i r s t extracellular microbial poly saccharides to be produced and used industrially. A comprehensive review (6) was published i n 1966 on dextran production, 1/

^References other than reviews, which c i t e o r i g i n a l research publications not included here, are marked with an asterisk. 284


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structure, properties, uses, and related considerations. The same topics were reviewed from a different viewpoint i n 1973 (7). The biosynthesis and structure of dextrans was reviewed comprehensively i n 1974 as w e l l as the s t r u c t u r a l l y dependent s p e c i f i c interactions with immunoglobulins (antibodies) and globulins such as concanavalin A (8). An extensive bibliography on a l l s c i e n t i f i c aspects of dextran (exclusive of c l i n i c a l research and testing) and dextran derivatives includes information from 1861 through mid-1976 (9). Summarized here i s the current status of dextran as an established product of world commerce and i n r e l a t i o n to s p e c i f i c industries. Interest i n p u l l u l a n and i t s p r a c t i c a l p o t e n t i a l i t i e s have developed since 1959 when Bender, Lehman, and Wallenfels (10) f i r s t characterized the water-soluble e x t r a c e l l u l a r product from Aureobasidium (Pullularia) pullulans and named i t accordingly^ The polysaccharide had been isolated previously and p a r t i a l l y characterized i n studies of micro organisms responsible f o r breakdown of forest l i t t e r (11). The slime-forming black yeastlike fungus, A. pullulans, occurs ubiquitously i n organic waste matter which i t decomposes i n s o i l , r i v e r s , paper-mill effluents, and sewage (12). TTie microorganism has adverse economic importance because of i t s costly deterioration of paint, discoloration of lumber, and attack on plants and plant products (13). In none of these natural occurrences, however, does the polysaccharide seem to have a role except as a slimy nuisance. Already of applied p r a c t i c a l l y , however, i s the enzyme pullulanase (pullulan 6-glucanohydrolase EC 3.2.1.41) which was discovered by Bender and Wallenfels i n Aerobacter aerogenes (14) and shown to depolymerize p u l l u l a n to i t s repeating unit maltotriose by s p e c i f i c attack on the i n t e r u n i t α-1,6-linkages. Pullulanase, now obtainable i n p r a c t i c a l amounts from numerous microbial sources, also cleaves α-1,6-linkages i n starch and i s used i n d u s t r i a l l y to release the unit chains i n starch (15,16). Substrates other than p u l l u l a n , however, may be use3~for producing pullulanase. The production, properties, and potential uses of p u l l u l a n have been reviewed (12). Summarized here are the constitutional bases for p r a c t i c a l applications and the uses that have been proposed. Importance of Naturally Occurring Dextrans The p r e d i l e c t i o n of Leuconostocs for sucrose i n nature has a s p e c i f i c basis. Sucrose induces i n these bacteria formation of the dextran-synthesizing enzyme dextransucrase (sucrose: 1,6-a-g-glucan 6-a-glucosyltransferase, E.C. 2.4.1.5). This enzyme accomplishes dextran synthesis by


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transferase action without need for intermediate substrates. Fructose, the byproduct of dextran synthesis, i s metabolized by Leuconostocs which cannot, however, metabolize either sucrose (they have neither invertase nor sucrose phosphorylase) or dextran. Extracellular dextransucrase i s produced abundantly by many s t r a i n s , although the dextransucrase of some strains i s cell-bound. The rate and extent of a c t i v i t y on sucrose that may result i s i l l u s t r a t e d dramatically by an h i s t o r i c report of the fortuitous conversion of 5000 l i t e r s of molasses to a compact gel mass i n 12 hours (4). In 1972, the status of the situation was tKat, "although i t i s d i f f i c u l t to quantify the effects of polysaccharides on the economics of sugar cane processing, i t i s obvious from the volume of recent l i t e r a t u r e that importance i s attached to t h e i r elimination from the process" (17). The dextrans have a major role (£,17) although b a c t e r i a l levans and polysaccharides of plant o r i g i n are involved also (17). The long-known adverse effects of dextran continue i n p o l a r i z a t i o n measurements, c l a r i f i c a t i o n and f i l t r a t i o n , and i n reducing the rate and efficiency of c r y s t a l l i z a t i o n . In addition, traces of dextran cause i n f e r i o r c r y s t a l structure by elongating the c axis (18,19). The beet sugar industry i s less affected by dextran contamination. Sucrose i s less exposed to infection during harvesting and f i r s t stages of processing of beets than of cane. Very sensitive biochemical tests have demonstrated the extent of dextran contamination i n commercial sucrose, including that distributed as a standard of highest purity. N e i l l , Hehre, and coworkers (20) demonstrated serological a c t i v i t y indicative of dextran i n both cane and beet sugars from diverse geographical sources and various methods of manufacture. The majority of the cane products showed higher serological a c t i v i t y than did the majority of the beet products. The weakest a c t i v i t i e s were i n several sanples of reagent grade sucrose of German o r i g i n prepared from beet sugar. Gibbons and Fitzgerald (21), u t i l i z i n g the agglutinizing action of dextran on c e l l s oF"Streptococcus mut ans, also demonstrated dextran i n reagent-grade sucrose. Dextranases are being investigated (22) and used (23) for removal of dextran from cane sugar juices as w e l l as from sucrose solutions and wines made hazy by the presence of dextran. Constitutional Basis f o r P r a c t i c a l Importance Dextran. By d e f i n i t i o n , the generic name dextran applies to a large class of α-D-glucans i n which predominance of a-l,6-linkages i s the common feature. One of the simplest dextrans known i s that from Leuconostoc mesenteroides NRRL B-512(F); the structural features are shown i n Figure 1.


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The α-y-glucopyranosidic linkages are 95% 1,6-and 5% 1,3(24). The 1,3-linkages are points of attachment of side drains of which about 85% are 1 or 2 glucose residues i n length (25). The remaining 15% of the side chains may have an average length of 33% glucose residues and may not be uniformly distributed i n the macromolecule (26). This dextran i s readily soluble i n water; certain other dextrans may be insoluble. In dextrans from other strains, the non1,6-linkages may be 1,2-, 1,3-, or 1,4-. Only one type may occur i n a dextran, or there may be two or three. Great d i v e r s i t y i s thus created. Dextran available i n the United States and western Europe i s produced from sucrose by s t r a i n NRRL B-512(F). Dextrans of apparently similar structure, but from different s t r a i n s , are produced i n Japan (27) and Russia (28). Dextrans produced i n other countries ÔT Europe and Asia are from selected strains (29*). Dextran having t h i s structure (Figure 1) was selected for production because the fraction (R^ 75,000 +_ 25,000) prepared from i t f o r intravenous administration (blood volume expander) was substantially less antigenic as compared with that from dextrans having higher percentages of non1,6-linkages. Dextran from s t r a i n NRRL B-512(F) i s completely metabolized i n man (30) when either ingested or administered parenterally as a fraction of suitable molecular size and size d i s t r i b u t i o n . Deri vat i z a t ion, however, slows or i n h i b i t s metabolism. An additional asset of s t r a i n NRRL B-512(F) i s i t s copious formation of dextransucrase (31). Production of dextran by use o f c e l l - f r e e culture f i l t r a t e s rather than i n growing cultures results i n enhanced y i e l d , quality and ease of p u r i f i c a t i o n o f the product. And furthermore, by suitable adjustment o f conditions, the major product can be synthesized d i r e c t l y within a chosen molecular weight range (32, 9). The native dextran may have weight-average molecular weight ( F y values (33) (light scattering) of 35-50 X 10 . The structural s i m p l i c i t y of this dextran permits graded p a r t i a l depolymerization and separation into fractions of any desired î% and size d i s t r i b u t i o n , which d i f f e r primarily i n molecular weight. The content of branch points remaining, however, would depend on the method of p a r t i a l depolymerization; i t i s decreased by acid hydrolysis (34) but not by use of endo-acting dextranases (1,6-a-g-glucan 6-glucanohydrolase, E.C. 3.2.1.11). Fractions o f lower molecular weight obtained through such enzymolysis r e t a i n the branch points, and t h e i r structural details would be determined by the action pattern of the s p e c i f i c dextranase (35). The series of fractions produced from p a r t i a l depolymerizates i s unique. Selected fractions or derivatives o f them serve pharmaceutical or other purposes having s p e c i f i c requirements f o r molecular

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size i n order to achieve physiological compatibility or other special objectives. Production of such fractions by d i r e c t , controlled enzymatic synthesis i s not known to be i n use. The high proportion of 1,6-linkages i n dextran NRRL B512(F) confers unusual f l e x i b i l i t y on the chain and leaves numerous s i t e s for substitution, essentially a l l of which are i n secondary positions. The r a t i o of r e l a t i v e rate constants established f o r methylation of the hydroxyl groups, C :C :C^:8:1:3.5 (36) indicates also the r e l a t i v e r e a c t i v i t y towards other substituents such as the sulfate (37). The frequent occurrence o f three hydroxyl groups i n consecutive positions i n the glucopyranosidic residues of dextrans would appear to account f o r t h e i r unusual a b i l i t y to complex with large amounts o f metallic elements such as f e r r i c iron and calcium. Such complexes are important as pharmaceutical preparations and i n certain metallurgical processes. Thus, the charcteristics of dextran from s t r a i n NRRL B512(F) that determine i t s value i n p r a c t i c a l applications, reside i n i t s composition as a soluble α-D-glucan and i n the properties o f i t s primary structure. In contrast, i t has been emphasized i n t h i s symposium that the unique characteristics of the anionic heteropolysaccharide xanthan which are basic to i t s usefulness, result from secondary and t e r t i a r y structural effects (58,39,40). The s p e c i f i c role of ionic charge, which also may Be" i n f l u e n t i a l i n xanthan properties, has not been established but may be inferred from research on ionogenic derivatives o f dextran (41). Pullulan. The generic name pullulan i s applied to any extracellular α-g-glucan elaborated by A. pullulans from a variety of substrates. A commonly observed feature i s the predominant repeat unit maltotriose polymerized l i n e a r l y through 1,6-linkages (Figure 2). Frequently present also are α-maltotetraose units (42,43,44) contained mainly w i t h i n the polymer chain (43J m amounts of 6.61 (43) and 5-7% (44). In products in~which possible heterogeneity was not excluded, traces of other neutral sugars and uronic acids have been reported (12). Products from other strains and from other genera and species have shown variation on the basic pullulan pattern such as the presence of 1,5linked glucosyl residues (45,46). Thus, "there i s , perhaps, no unique structure of pulTATlin (45). The molecular weight of a pullulan product d i f f e r s with the lengtja o f fermentation time (47,48). Molecular weight of 2 X 10 developed ^ n i t i a l l y during limited fermentation decreased to 1.5 X 10 during continued fermentation (47). The s i t e of degradation i s the i n t e r n a l l y located maltotetraose u n i t s ; the degradative enzyme appears to be an "endoamylase" produced during culture growth (45^47). The modified 2



3

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p u l l u l a n resulting from "endoamylase" action i s inert to aamylase (43). Such uncontrolled v a r i a t i o n i n molecular weight can be eliminated, however, by choice of s t r a i n and adjustment of the pH and of the phosphate content of the culture medium (49). Pullulan products having molecular weights as high as 2ζΰ X 10 or as low as 5 X 10 may be obtained i n t h i s way. The mechanism of biosynthesis of pullulan discourages consideration of enzymatic synthesis as a means f o r production. Synthesis i s accomplished through mediation of sugar nucleotide/ l i p o i d c a r r i e r intermediates associated with c e l l membrane fractions (50). The pullulan molecule may be considered as a chain of amy lose, the l i n e a r component of starch, i n which an a-1,6bond replaces every t h i r d a-l,4-bond. The 1,6-bond introduces f l e x i b i l i t y , and the interrruption of regularity results i n making pullulan readily soluble, eliminating rétrogradation and improving rather than impairing f i b e r - and film-forming a b i l i t y . The presence of the 1,6-bonds may influence the p o s i t i o n of substituents and properties of derivatives by introducing a different sequence of free hydroxyl groups. The presence of 1,6-linkages, spaced as they are, prevents attack by salivary and i n t e s t i n a l amylases (43). Isoamylase from Pseudomonas sp., which cleaves a-l,6-boncEs i n amylopectin and glycogen, also i s inert on pullulan (51). Dextran and Dextran Derivatives i n Industry Pharmaceutical Industry. Probably the largest outlet for dextran and dextran derivatives i s through the pharmaceutical and fine chemicals industries. The major developments have originated from fundamental research i n Sweden which was i n i t i a t e d about 1944 and has continued consistently (52,53). Research and development have followed, however, i n numerous other countries throughout the world which produce t h e i r own pharmaceutical products from dextran (9,27,28,29*). Two dextran fractions of major significance are used i n suitably prepared solutions f o r parenteral administration ((^,9). The fraction of M 70,000 i s used to restore and maintain blood volume i n treatment of shock, hemorrhage, extensive burns,_and a variety of other physiological conditions. The f r a c t i o n of M 40,000 i s used to improve flow i n c a p i l l a r i e s , treatment of vascular occlusion, a r t i f i c i a l extracorporeal perfusion of organs, and i n a variety of other ways. These and other sharply cut dextran fractions are used for preparation of numerous derivatives such as the sulfates, diethylaminoethyl (DEAE) dextran, and complexes with iron and other metallic elements. These substances serve a variety of purposes (£,54,55). Dextran sulfates have anticoagulant, antilipemic, and antiulcer a c t i v i t y . They w


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are used i n l i q u i d two-phase s e p a r a t i o n and c o n c e n t r a t i o n o f l i v i n g c e l l s such as those o f v i r u s e s , b l o o d , tumors, and other t i s s u e s . DEAE d e x t r a n enhances b i o l o g i c a l e f f e c t s o f macromolecules and v a c c i n e s . A s o l u b l e complex o f dextran and i r o n i s produced w i d e l y i n numerous c o u n t r i e s f o r i n t r a muscular a d m i n i s t r a t i o n t o a l l e v i a t e i r o n - d e f i c i e n c y anemia i n the human and i n domestic animals. The s o l u t i o n contains 5% i r o n and 20% dextran o f M 5,000 (56). The i r o n i s m a i n l y n o n i o n i c (56) and appears t o be B-FeOOH (57). The i n i t i a l patents Ç5ÏÏ) have been emulated e x t e n s i v e l y ( £ ) . A s o l u b l e c a l c i u m complex c o n t a i n i n g 10-121 c a l c i u m i s administered p a r e n t e r a l l y t o a l l e v i a t e hypocalcemia o f c a t t l e d e l i v e r y p a r e s i s (9). Complexes w i t h antimony and a r s e n i c are e f f e c t i v e a g a i n s t t r o p i c a l i n f e c t i o n s (9). C r o s s l i n k e d dextran g e l s o r t h e i r i o n i c d e r i v a t i v e s are employed i n p u r i f i c a t i o n , f r a c t i o n a t i o n and i s o l a t i o n o f enzymes, hormones, and o t h e r s e n s i t i v e b i o l o g i c a l substances w i t h o u t m o d i f i c a t i o n o f t h e i r a c t i v i t y . By covalent bonding t o e i t h e r dextran o r c r o s s l i n k e d dextran g e l s , enzymes, immunoglobulins, and antigens are s t a b i l i z e d and supported f o r use i n s p e c i f i c r e a c t i o n s ( 5 9 , 9 ) . Dextranases, prepared by growth o f v a r i o u s molds on d e x t r a n s , are used i n mouthwashes and toothpaste t o e i t h e r d i s p e r s e o r i n h i b i t f o r m a t i o n o f d e n t o - b a c t e r i a l plaques which c o n t a i n dextrans and f o s t e r c a r i o u s d e n t a l l e s i o n s (9,60,61,62). TFoodTndustry. The p o t e n t i a l i t i e s f o r dextran i n the food i n d u s t r y have been reviewed (63^, 64). The o n l y a c t u a l uses known t o the a u t h o r , however, are i n dextran g e l f i l t r a t i o n processes t o concentrate p r o t e i n s o r t o recover p r o t e i n s from l i q u i d wastes and e f f l u e n t streams. From c e r e a l waste streams, 70% recovery o f p r o t e i n has been effected. In the m i l k i n d u s t r y , skim m i l k or cheese whey i s f r a c t i o n a t e d f o r recovery o f undenatured p r o t e i n components o f enhanced q u a l i t y , n u t r i t i v e v a l u e , and a p p l i c a b i l i t y . A p l a n t having c a p a c i t y o f 1 X 10 l b . per day i s i n o p e r a t i o n (65). P r o t e i n i s separated from l a c t o s e and m i n e r a l c o n s t i t u e n t s ancT f r a c t i o n a t e d m a i n l y on the b a s i s o f molecular weight i n t o c a s e i n , β - l a c t o g l o b u l i n , and α - l a c t o g l o b u l i n . βL a c t o g l o b u l i n , which i s h i g h l y s u p e r i o r n u t r i t i o n a l l y t o c a s e i n (66), had r e s t r i c t e d use when p r e v i o u s l y i s o l a t e d as the degraded and denatured l a c t a l b u m i n (67). Other v a l u a b l e products t h a t may be recovered are l a c t o T e r r i n and immuno globulins. By another a p p l i c a t i o n o f g e l f i l t r a t i o n , the p r o t e i n content o f m i l k i s i n c r e a s e d from 3.351 t o 5.35% w i t h o u t i n c r e a s e o f the l a c t o s e and m i n e r a l contents (68). Atomic F u e l and M e t a l l u r g y . G e l p r e c i p i t a t i o n i s a process i n which dextran (or c e r t a i n other p o l y h y d r i c polymers)


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i s used to produce a metal compound i n the form of a gel under conditions where an insoluble precipitate would be expected (69) (Figure 3). The process i s used for purifying, separating, and concentrating metals from solutions of t h e i r salts or mixtures of salts or from c o l l o i d a l dispersions of. aqueous hydrous sols. The f i n a l product may be i n the form of powder, granules, spheres, rods or shaped rods, or ceramic coatings and moulded objects. Products prepared by use of dextran as the g e l l i n g agent are f o r use as nuclear reactor fuels (70,71), catalysts (71,72,73), ceramic coatings (70), refractories and f e r r o - e l e c t r i c materials Ç72,73), and powder f o r alloys (72), pigments (74) and metallurgical processes (70). The g e l l i n g agent and metal ions appear to form molecular complexes which, when contacted with the precipitating reagent, produce discrete macrocrystalline gel p a r t i c l e s (69,75). The 1,6-linkages i n dextran are believed to confer a special configuration on the three contiguous free -OH groups which i s p e c u l i a r l y favorable to -OH--complex formation with an unusual number of metal ions (6£, 76). The s p e c i f i c properties of dextran metallic ion complexes are u t i l i z e d i n separating f e r r i c iron from mixtures with copper, n i c k e l or cobalt, or n i c k e l from thorium, or zirconium from copper (76). Dextran (or fractions of stated molecular weight) i s u t i l i z e d i n preparing black magnetic i r o n oxide (Fe^O.) from ferrous s a l t (74,77). Cupric ion may be adsorbed from solution on hydrous gels" ÔT f e r r i c oxide, chromium oxide, or thorium phosphate/dextran g e l , and then eluted (ZI). The procedures reviewed here indicate the potential for dextran application i n gel-precipitation processes. Some of the procedures are known to be i n use. Petroleum Production. A pioneering concept advocated for some years was to make dextran a profitable byproduct of the sugar cane industry by using i t i n petroleum d r i l l i n g muds (78). In i n i t i a l laboratory testing f o r water loss i n h i b i t i o n , a modified dextran gave results equivalent or superior to starch and carboxymethyl cellulose (79). The modified dextran (Viscoba), prepared by treatment of dextran with aldehyde before i s o l a t i o n (80), had improved v i s c o s i t y and was resistant to microbial attack. The concept was advanced further when, during 1956 through 1959, a dextran production p i l o t plant was operated i n conjunction with a sugar m i l l i n Cuba (81). The dextran, produced from s t r a i n NRRL B-512(F) by a modified enzymatic procedure, was precipitated once and drum dried. [The fructose byproduct was recovered and uses investigated (81)]. The output (3-6 tons/day) was used i n the United States i n d r i l l i n g muds under a variety of f i e l d conditions. The price at the well s i t e was 46 cents/lb. ; the demand greatly exceeded the supply (82). The dextran


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Figure

1.

Structural features of dextran from Leuconostoc mesenteroides NRRL B-512(F)

Figure 2. The characteristic structural features of pullulans: a-maltotriose polymerized through a-lfi-lirikages

Solution

(acidic)

P r e c i p i t a t i n g Reagent

M e t a l l i c salt Gelling agent

NaOH or NH4OH (Dextran)

Solution

solution,

NH 3 g a s , or amines

Gel

Mixed

Non-coalescent,

Reduce

Non-adherring,

alloy

Mixed Gelling

[acidic)

metallic

salts

agent

Gel

Separated

to

Components

powder

Microcrystalline Dry:

to p a r t i c l e s of

desired shape and size Oxides:

oxidize

Oxides,

hydroxides:

gas to m e t a l

Figure 3.

by air reduce by

powders

The gel-precipitation process and some of the products resulting


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functioned better than some ot i t s competitors and as well as any.— The dextran could not be used i n lime base muds; i t precipitated at pH 11.0-11.2 and l o s t i t s water-binding capacity. Under these conditions, however, l i g h t l y hydroxy ethylated dextran retained i t s water-binding capacity (82). At less basic or neutral pH, dextran tolerates calcium ion and magnesium ion w e l l (83). Dextran i s the hydrocolloid i n an "inhibited mud" composition containing 3500 ppm calcium ion that i s used to i n h i b i t shale hydration (84). Like a l l polymers, dextran i s susceptible to free r a d i c a l degradation, and protection i s advised during processing as well as use (85)· Dextran NRRL B-512(F) has properties suitable f o r use i n viscous water flooding (86); i n screening tests, i t gave results superior to many otEêr substances examined. The unfavorable results of a f i e l d t r i a l (87) may have related to lack of protection against free-radical degradation. Photographic Industry. Native high molecular weight dextran has been supplied consistently for an undisclosed i n d u s t r i a l use believed to relate to photographic products. Numerous patents have been issued i n the past and continue to be issued on the superior effects achieved from certain dextran derivatives i n X-ray and photographic emulsions (9^54,55). I t seems probable that some of these derivatives are i n use. Pullulan--Proposed Uses Numerous applications o f pullulan and i t s derivatives have been proposed and patented, but apparently are not yet i n use (88,89). The p o s s i b i l i t y of eventual success for most uses i s increased by the claim that, by proper selection of pullulan-producing s t r a i n s , the molecular weight can be controlled and absence of black pigment i n the product can be assured (49). The efficiency of pullulan, even as the crude fermentation liquors, has been demonstrated for flocculation of clay slimes from aqueous solutions resulting from beneficiation of uranium, potash, and other ores (90,91,92). Films formed from pullulan without~plasticizers have excellent physical properties, are water-soluble, impervious to oxygen and suitable f o r coating or packaging foods and pharmaceuticals especially when exclusion o f oxygen i s desirable (88). Fibers from pullulan have a shiny gloss and high t e n s i l e strength which, after stretching, i s described — Death of the key personnel i n an airplane accident terminated this development.

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as comparable to that of nylon (88). The fibers may be admixed with natural fibers i n special papers and other products (93). Pullulan i s suitable for making adhesives and shaped a r t i c l e s by compression molding. In such molded a r t i c l e s pullulan has characteristics similar to polyvinyl alcohol or to styrene (88). I t has desirable properties for use i n noncaloric and other foods; i t i s nontoxic and nondigestible (88). Pullulan i s biodegradable, however, under usual conditions of waste disposal.

Literature Cited 1. 2. 3. 4. 5. 6. 7.

8. 9.

10. 11. 12.

13. 14. 15. 16.

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